The present invention relates to regulatory elements capable of mediating the translational efficiency of a transcript linked in operative association therewith. The translational regulatory elements of the present invention may also be used in combination with other regulatory elements, such as promoters, enhancers, or fragments thereof, to modify the levels of expression of a gene of interest within a host
Regulatory elements within the 5xe2x80x2 and 3xe2x80x2 untranslated regions (UTR) of genes are known to mediate transcriptional and translational efficiencies of associated promoters and transcripts, respectively. These regulatory elements can modulate gene expression in an organism through a number of mechanisms including transcription, translation, and RNA stability. For example, some 5xe2x80x2 non-translated regions (5xe2x80x2 leaders) are known to enhance the translational efficiency of mRNA, resulting in an increased accumulation of recombinant protein many fold.
Some of these 5xe2x80x2 leaders affect gene expression by quantitative enhancement of transcription, as with the UTR of the thylakoid protein genes PsaF, PerH and PetE from pea (Bolle et al., 199, Plant J. 6, 513-523), or by repression of transcription, as for the 5xe2x80x2 UTR of the pollen-specific LAT59 gene from tomato (Curie and McCormick, 1997, Plant Cell 9, 2025-2036). Some 3xe2x80x2 regulatory regions contain sequences that act as mRNA instability determinants, such as the DST element in the Small Auxin-Up RNA (SAUR) genes of soybean and Arabidopisis (Newman et al., 1993, Plant Cell 5, 701-714).
Other 5xe2x80x2 leaders have been shown to contain translational enhancer sequences or structures such as the Omega sequence of the 5xe2x80x2 leader of the tobacco mosaic virus (Gallie and Walbot, 1992, Nucleic Acid Res. 20, 4631-4638), the 5xe2x80x2 alpha-beta leader of the potato virus X (Tomashevskaya et al, 1993, J. Gen. Virol. 74, 2717-2724), and the 5xe2x80x2 leader of the photosystem I gene psaDb of Nicotiana sylvestris (Yamamoto et al., 1995, J. Biol. Chem 270, 12466-12470). In plants, most of the translation enhancers characterized belong to plant virus 5xe2x80x2 leaders (Gallie, D. R., et al., 1987, NAR 15, 8693-8711.; Jobling and Gehrke, 1987, Nature 325, 622-625; Gehrke, 1989, U.S. Pat. No. 4,820,639.; Smimyagina, et al 1991, Biochimie 73: 998-1011; Wilson, 1996, U.S. Pat. No. 5,489,527). Heat shock genes have been shown to contain a sequence that enhances translation in their 5xe2x80x2 non-translated leader in animals and plants (McGarry and Lindquist, 1985 Cell 42, 903-911; Austin, 1994 U.S. Pat. No. 5,362,865). Some other plant 5xe2x80x2 non-translated leaders have also been shown to enhance translation under normal (Yamamoto et al, 1995) and specific stress conditions (Pitto et al, 1992, Plant Physiol. 100; 1827-1833; Bailey-Serres and Dawe, 1996, Plant Physiol. 112: 685-695). Other translational enhancers are also known in the literature (e.g. Helliwell and Gray 1995, Plant Mol. Bio. vol 29, pp. 621-626; Dickey L. F. al. 1998, Plant Cell vol 10, 475-484; Dunker B. P. et al. 1997 Mol. Gen. Genet. vol 254, pp. 291-296). These translational regulatory elements are directed to enhancing translational activities, and have relatively low efficiencies. Synthetic random sequences used as 5xe2x80x2 non-translased leaders are considered to have a neutral effect on translation efficiency and are used as reference points for minimum translation in studies of translation enhancers (Datla et al, 1993,Plant Science 94:139-149; Bates et al. 1996, Plant. J. 10: 613-623).
The present invention is directed to translational regulatory elements that mediate the translation efficiency of a transcript in operative association therewith by enhancing or inhibiting translation efficiency. These translational regulatory elements are equally, or more, active than those of the prior art. The translational regulatory elements of the present invention may be used to mediate the translation efficiency of chimeric transcripts comprising one or more translational regulatory elements. Furthermore, the translational regulatory elements of the present invention may be used within chimeric constricts comprising other regulatory elements, for example, promoter, enhancer, silencer, or other elements, or a combination thereof.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.
The present invention relates to regulatory elements capable of mediating the translational efficiency of a transcript linked in operative association therewith. The translational regulatory elements of the present invention may also be used in combination with other regulatory elements, such as promoters, enhancers, or fragments thereof, to modify the levels of expression of a gene of interest within a host.
According to the present invention there is provided an isolated nucleic acid comprising the nucleotide sequence defined by SEQ ED NO:6, or a fragment, derivative or analog thereof, wherein said fragment, derivative or analog thereof exhibits translational regulatory activity. Also included within the present invention is a construct comprising, at least one isolated nucleic acid as just defined, in operative association with a gene of interest, and one or more regulatory elements required for the expression of the gene of interest within a host organism. Preferably, the one or more regulatory elements comprise a regulatory element selected from the group consisting of an inducible promoter, developmentally regulated promoter, tissue specific promoter, constitutive promoter, and enhancer element.
This invention also relates to a method of increasing the amount of protein produced in an organism comprising, transforming the organism with a construct as defined above, growing the organism, and obtaining the protein therefrom.
Furthermore, the present invention relates to an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 6, 7, 8, 9, and a fragment, derivative, analog or a combination of a fragment, derivative or analogue, of the nucleotide sequence selected from the group consisting of SEQ ID NO:5, 6, 7, 8 and 9, wherein the fragment, derivative analog, or combination thereof exhibits translational regulatory activity. The present invention also includes a construct comprising, at least one isolated nucleic acid as just defined, in operative association with a gene of interest, and one or more regulatory elements required for the expression of the gene of interest within a host organism. Preferably, the one or more regulatory elements comprise a regulatory element selected from the group consisting of an inducible promoter, developmentally regulated promoter, tissue specific promoter, constitutive promoter, and enhancer element.
The present invention also pertains to transgenic hosts comprising the constructs as defined above. The transgenic host are selected from the group consisting of a plant, tree, animal, insect, yeast and bacteria. Preferably, the transgenic host is a plant.
The present invention also embraces a method of mediating the translational activity of a transcript comprising, transforming a host with any one construct as defined above, and growing the host.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.